Analysis of Apoptosis by Flow-cytometry

Analysis of Apoptosis by Flow-cytometry Apoptosis Changes in morphology Alteration of membrane integrity Cell shrinking Chromatin condensation PS exposure Drop in ∆ψ m Membrane alteration caspase activation Cell fragmentation Chromatin condensation DNA fragmentation Apoptotic bodies and phagocytosis Alteration of Morphology Granularity Living cells Late apoptotic Early apoptotic Thymocytes Dead cells 24h DXM FACS acquisition Size Membrane integrity Staining of apoptotic cells with : YO-PRO-1 7-AAD Staining of end staged dead cells with PI Membrane integrity- Costaining with YO-PRO-1 and PI Treated PI Untreated YO-PRO-1 Jurkat cells 4hrs Camptothecin 10mM Co-staining with YO-PRO-1 and PI Phosphatidylserine exposure PS, normally located on the cytoplasmic surface of the cell membrane, becomes exposed to the extracellular environment. Annexin V (human vascular anticoagulant with high affinity for PS), allows the detection of PS. PI Jurkat + camptothecin Annexin-V Two Examples (Left and Right) of Triple Stained (HO-AnnexFITC and PI) DHD Cells UV light Blue light Broken nucleus Intact cells are blue, apoptotic cells are green, late apoptotic necrotic cells are multilabelled. Two Major Apoptotic Pathways in Mammalian Cells Mitochondrial pathway Death receptor pathway DNA damage CD95L CD95 p53 DyM Bcl-2 FADD c-FLIP Bcl-xl Bax Procaspase-8 Bid caspase-8 Caspase-8 Procaspase-3 truncated bid cyto c Apoptosome AIF Apaf-1 Pro-casp-9 Type I cells Type II cells caspase-3 IAPs Smac/DIABLO Apoptotic substrates Caspase activation Jurkat cells 4hrs Camptothecin 10mM Co-staining with FLICA and PI FLICA: caspase inhibitor, binds to the reactive cysteins on active caspases and inhibits further enzymatic activity. Unbound FLICA diffuses out of the cell and is washed away; The green fluorescent signal measures the amount of active caspase that was present at the time FLICA was added. Detection of active caspase-8 PI Untreated Jurkat cells PI+, caspase- (Dead) Camptothecin-treated cells PI-, caspase+ (Apoptotic) Caspase-8 reagent Two Major Apoptotic Pathways in Mammalian Cells Mitochondrial pathway Death receptor pathway DNA damage CD95L CD95 ∆ψ DyM m p53 Bcl-2 FADD c-FLIP Bcl-xl Bax Procaspase-8 Bid caspase-8 Caspase-8 Procaspase-3 truncated bid cyto c Apoptosome AIF Apaf-1 Pro-casp-9 Type I cells Type II cells caspase-3 IAPs Smac/DIABLO Apoptotic substrates Mitochondrial Structure and Function ∆ψ m assessment using DIOC 6(3) and JC-1 dyes: DIOC6(3) accumulates in the mitochondrial matrix under the influence of the ∆ψ m JC-1 forms aggregates under high mitoch ∆ψ m, which fluoresce in red, whereas the monomeric form fluoresces in green. Mitochondrial structure analysis:Nonyl Acridine Orange incorporation. Reveals alteration of cardiolipids in mitoch membrane. 1 cardiolipid molecule binds 2 NAO molecules=> decreased fluorescence means decreased or altered cardiolipids. Thymocytes stained with JC1 following incubation : Living 4 C 37 C DXM 1µM Apoptotic Kinetics of mitochondrial alterations in thymocytes treated with DXM DIOC6(3) NAO DNA fragmentation Confocal microscopy analysis of apoptotic thymocytes JC-1 DIOC6(3) J-aggregates Monomeres Extinction of mito NAO Fluo/PI+ nuclei PI/NAO Living thymocytes Apoptotic thymocytes Time course analysis of early events in DXM-apoptosis ∆ψ m Fragmented DNA Trypan blue staining